Improvement of Trehalose Production by Immobilized Trehalose Synthase from Thermus thermophilus HB27.

Trehalose is a non-reducing disaccharide with a wide range of applications in the fields of food, cosmetics, and pharmaceuticals. In this study, trehalose synthase derived from Thermus thermophilus HB27 (TtTreS) was immobilized on silicalite-1-based material for trehalose production. The activity and the stability of TtTreS against pH and temperature were significantly improved by immobilization. Enzyme immobilization also led to a lower concentration of byproduct glucose, which reduces byproduct inhibition of TtTreS. The immobilized TtTreS still retained 81% of its initial trehalose yield after 22 cycles of enzymatic reactions. The immobilized TtTreS exhibited high operational stability and remarkable reusability, indicating that it is promising for industrial applications.

Generation of a platform strain for ionic liquid tolerance using adaptive laboratory evolution.

BACKGROUND: There is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass. METHODS: To generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40 days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing. RESULTS: The most prevalent mutations in tolerant clones occurred in transport processes related to the functions of mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations (i.e., cross tolerance) with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc]. CONCLUSION: The generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering.

Metabolic responses in Candida tropicalis to complex inhibitors during xylitol bioconversion.

During xylitol fermentation, Candida tropicalis is often inhibited by inhibitors in hemicellulose hydrolysate. The mechanisms involved in the metabolic responses to inhibitor stress and the resistances to inhibitors are still not clear. To understand the inhibition mechanisms and the metabolic responses to inhibitors, a GC/MS-based metabolomics approach was performed on C. tropicalis treated with and without complex inhibitors (CI, including furfural, phenol and acetic acid). Partial least squares discriminant analysis was used to determine the metabolic variability between CI-treated groups and control groups, and 25 metabolites were identified as possible entities responsible for the discrimination caused by inhibitors. We found that xylose uptake rate and xylitol oxidation rate were promoted by CI treatment. Metabolomics analysis showed that the flux from xylulose to pentose phosphate pathway increased, and tricarboxylic acid cycle was disturbed by CI. Moreover, the changes in levels of 1,3-propanediol, trehalose, saturated fatty acids and amino acids showed different mechanisms involved in metabolic responses to inhibitor stress. The increase of 1,3-propanediol was considered to be correlated with regulating redox balance and osmoregulation. The increase of trehalose might play a role in protein stabilization and cellular membranes protection. Saturated fatty acids could cause the decrease of membrane fluidity and make the plasma membrane rigid to maintain the integrity of plasma membrane. The deeper understanding of the inhibition mechanisms and the metabolic responses to inhibitors will provide us with more information on the metabolism regulation during xylitol bioconversion and the construction of industrial strains with inhibitor tolerance for better utilization of bioresource.

Strategies for enhancing microbial tolerance to inhibitors for biofuel production: A review.

Using lignocellulosic biomass for the production of renewable biofuel provides a sustainable and promising solution to the crisis of energy and environment. However, the processes of biomass pretreatment and biofuel fermentation bring a variety of inhibitors to microbial strains. These inhibitors repress microbial growth, decrease biofuel yields and increase fermentation costs. The production of biofuels from renewable lignocellulosic biomass relies on the development of tolerant and robust microbial strains. In recent years, the advancement of tolerance engineering and evolutionary engineering provides powerful platform for obtaining host strains with desired tolerance for further metabolic engineering of biofuel pathways. In this review, we summarized the inhibitors derived from biomass pretreatment and biofuel fermentation, the mechanisms of inhibitor toxicity, and the strategies for enhancing microbial tolerance.

Furfural tolerance and detoxification mechanism in Candida tropicalis.

BACKGROUND: Current biomass pretreatment by hydrothermal treatment (including acid hydrolysis, steam explosion, and high-temperature steaming) and ionic liquids generally generate inhibitors to the following fermentation process. Furfural is one of the typical inhibitors generated in hydrothermal treatment of biomass. Furfural could inhibit cell growth rate and decrease biofuel productivity of microbes. Candida tropicalis is a promising microbe for the production of biofuels and value-added chemicals using hemicellulose hydrolysate as carbon source. In this study, C. tropicalis showed a comparable ability of furfural tolerance during fermentation. We investigated the mechanism of C. tropicalis's robust tolerance to furfural and relevant metabolic responses to obtain more information for metabolic engineering of microbes for efficient lignocellulose fermentation. RESULTS: Candida tropicalis showed comparable intrinsic tolerance to furfural and a fast rate of furfural detoxification. C. tropicalis's half maximal inhibitory concentration for furfural with xylose as the sole carbon source was 3.69 g/L, which was higher than that of most wild-type microbes reported in the literature to our knowledge. Even though furfural prolonged the lag phase of C. tropicalis, the final biomass in the groups treated with 1 g/L furfural was slightly greater than that in the control groups. By real-time PCR analysis, we found that the expression of ADH1 in C. tropicalis (ctADH1) was induced by furfural and repressed by ethanol after furfural depletion. The expression of ctADH1 could be regulated by both furfural and ethanol. After the disruption of gene ctADH1, we found that C. tropicalis's furfural tolerance was weakened. To further confirm the function of ctADH1 and enhance Escherichia coli's furfural tolerance, ctADH1 was overexpressed in E. coli BL21 (DE3). The rate of furfural degradation in E. coli BL21 (DE3) with pET-ADH1 (high-copy plasmid) and pCS-ADH1 (medium-copy plasmid) was increased by 1.59-fold and 1.28-fold, respectively. CONCLUSIONS: Candida tropicalis was a robust strain with intrinsic tolerance to inhibitor furfural. The mechanism of furfural detoxification and metabolic responses were identified by multiple analyses. Alcohol dehydrogenase 1 was confirmed to be responsible for furfural detoxification. C. tropicalis showed a complex regulation system during furfural detoxification to minimize adverse effects caused by furfural. Furthermore, the mechanism we uncovered in this work was successfully applied to enhance E. coli's furfural tolerance by heterologous expression of ctADH1. The study provides deeper insights into strain modification for biofuel production by efficient lignocellulose fermentation.

Effects of acid impregnated steam explosion process on xylose recovery and enzymatic conversion of cellulose in corncob.

Corncob residue is a cellulose-rich byproduct obtained from industrial xylose production via dilute acid hydrolysis processes. Enzymatic hydrolysis of cellulose in acid hydrolysis residue of corncob (AHRC) is often less efficient without further pretreatment. In this work, the process characteristics of acid impregnated steam explosion were studied in conjunction with a dilute acid process, and their effects on physiochemical changes and enzymatic saccharification of corncob residue were compared. With the acid impregnated steam explosion process, both higher xylose recovery and higher cellulose conversion were obtained. The maximum conversion of cellulose in acid impregnated steam explosion residue of corncob (ASERC) reached 85.3%, which was 1.6 times higher than that of AHRC. Biomass compositional analysis showed similar cellulose and lignin content in ASERC and AHRC. XRD analysis demonstrated comparable crystallinity of ASERC and AHRC. The improved enzymatic hydrolysis efficiency was attributed to higher porosity in ASERC, measured by mercury porosimetry.